The long-term objective of this research project is to gain understanding of DNA replication at the molecular level. Frog virus 3 (FV3) replication in eukaryotic cells is used as a model system in meeting this objective. The structure of the molecular ends of the FV 3 genome will be determined utilizing the combined techniques of restriction endonuclease digestion and electron microscopy of DNA molecules. The fate of the parental genomes will be followed by electron microscopic autoradiography and equilibrium centrifugation in cesium chloride gradients. Site(s) of origin of FV 3 DNA replication will be determineed utilizing techniques of restriction endonuclease digestion and electron microscopy. The structure of replicating DNA molecules will be analyzed by the combined techniques of alkaline and neurtral sucrose gradients, restriction endonuclease digestion, and electron microscopy. I will isolate several DNA negative temperature-sensitive mutants by mutagenizing FV 3 with bromodeoxyuridine. These temperature-sensitive mutants will be used to determine the role of viral genes in the proposed two stages of FV 3 DNA replication. Methods to be used include standard virological, biochemical, and molecular biological ones and include gel electrophoresis, velocity and equilibrium centrifugation, and electron microscopy.